With recent unsettling events foreshadowing an increased sense of urgency, NIAID has targeted research and development of therapeutics, vaccines, adjuvants/immunostimulants, and diagnostics for smallpox (variola). TSRL was granted a Phase I SBIR to explore prodrug strategies to increase the oral availability of potential anti-smallpox drugs. Strategies that can improve the oral bioavailability of approved drugs as well as potential drug candidates will facilitate the development of more effective antiviral agents and reduce undesirable outcomes such as drug toxicity, poor patient compliance, and high costs. The long-term goal of this project for biodefense is to improve the oral absorption of potent, but poorly absorbed anti-pox agents by designing prodrugs targeted to transporters expressed in human intestine and able to be activated by endogenous esterases or amidases that release the absorbed drug in vivo from its prodrug form. For this project, we established collaborations with Prof. Charles McKenna (prodrug design and synthesis) at the University of Southern California and Prof. John Drach (virological studies) at the University of Michigan. The work in the Phase I project focused chiefly on prodrugs of cyclic cidofovir (cHPMPC), a broad spectrum antiviral compound with potent activity against variola and other orthopox viruses, but of limited value in a smallpox epidemic due to its very low oral bioavailability. We demonstrated that dipeptide prodrugs of cHPMPC, which were first discovered in Prof. McKenna's laboratory, have great potential for oral delivery. Our results from an animal oral bioavailability model indicate that absorption of the first generation of dipeptide prodrugs is up to an order of magnitude greater than that of the parent drug cHPMPC, and that a significant fraction of the absorbed prodrug is converted to drug in vivo. In this Phase II SBIR proposal, we have devised a synthetic strategy and SAR plan to optimize the bioavailability of this new class of prodrug. We have formed a collaborative network of researchers and specialists that will allow us to systematically test a diverse library of novel cHPMPC dipeptide conjugates for stability, transport, drug oral bioavailability, and efficacy in accepted animal models. The compound or compounds showing the best oral absorption and efficacy will be manufactured following GMP requirements and tested for toxicity. The combined portfolio of pharmacokinetic, metabolism, manufacturing and toxicological data will be used in support of an IND and future clinical testing. The long term objective of this project is to make new oral drugs for the treatment of small pox. The expertise needed to succeed in this project will be provided by personnel at TSRL, Inc. in collaboration with Dr. John Drach, an expert in the area of antiviral drugs at the University of Michigan, and Dr. Charles Mckenna at the University of Southern California, who is well experienced at organic and medicinal chemistry of phosphorous containing compounds.